Whisnant R E, Gilman A G, Dessauer C W
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6621-5. doi: 10.1073/pnas.93.13.6621.
Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by Gsalpha and forskolin is synergistic. These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme.
在大肠杆菌中独立合成腺苷酸环化酶的两个胞质结构域后，通过简单混合即可重建腺苷酸环化酶活性。我们已经合成并纯化了I型腺苷酸环化酶的C1a结构域和II型酶的C2结构域，以评估它们彼此之间以及与激活剂Gsα和福斯可林的相互作用。在没有激活剂的情况下，片段以低亲和力结合并显示出低催化活性。福斯可林或活化的Gsα均可将这种基础活性刺激100倍以上。此外，添加这些激活剂会增加片段彼此之间的表观亲和力。Gsα和福斯可林对催化的刺激是协同的。这些数据提示了一种模型，即Gsα或福斯可林可增强另一种激活剂与腺苷酸环化酶的结合，同时促进该酶C1和C2结构域之间的相互作用。
